Neurotransmitter receptors and ion channels in the central nervous system are localized to synaptic and extrasynaptic membrane compartments of pre- and postsynaptic elements of neurons. The impact of the activation of these proteins on synaptic integration and regulation of transmitter release depends on their precise location relative to synapses, as well as on the density and coupling of molecules in microcompartments of the cells. High-resolution qualitative and quantitative visualization of membranebound receptors and ion channels is, therefore, essential for understanding their roles in cell communication.

Molecular dynamics in the neuronal network

The ability of the nervous system to learn and respond to the environment reflects an underlying capability of neurons to dynamically alter the number, type, and strengths of their connections. These connections, called synapses, are highly organized sites of contact between postsynaptic neurons and presynaptic terminals. The specialized synaptic membrane, contains a large variety of molecules such as receptors, ion channels, and associated structural proteins, whose precise subcellular organization facilitates its proper function. A large number of studies have provided evidence that the location of these proteins and their position relative to synapses substantially affects their functional roles. Neurotransmitter receptors, localized to the synaptic membrane of postsynaptic compartments of neurons, are directly exposed to released neurotransmitters.

Consequently, they are activated in a transient manner, generating fast postsynaptic responses that are precisely time-locked to the presynaptic action potentials. In contrast, receptors localized to the extrasynaptic plasma membrane, remote from synaptic sites, are activated by spilledover neurotransmitters producing a tonic conductance that is not precisely time-locked to single presynaptic action potentials, but rather reflects the whole network activity on a slower time scale [1]. Receptors can also be located presynaptically either on the extrasynaptic membrane of axon terminals or over the presynaptic grid where they are activated by neurotransmitters released by the same or by neighbouring boutons. Like neurotransmitter receptors, ion channels are also localized to the somato-dendritic membranes and axon terminals of neurons. Postsynaptic channels are generally playing a role in the integration and plasticity of synaptic inputs, as well as in the control of neuronal excitation by mediating slow inhibitory synaptic responses and contributing to the resting membrane potential.

Presynaptic channels that are concentrated either at the presynaptic active zone or localized to the extrasynaptic membrane of boutons are involved in the regulation of neurotransmitter release, thereby playing a role in the presynaptic modulation of neuronal activity. It is, therefore, easy to understand that the same receptor and ion channel could fulfill very different functional requirements when targeted to different subcellular compartments of cells [1].

Furthermore, the impact of the activation of membrane proteins on synaptic integration and regulation of transmitter release critically depends on the density and functional coupling of receptors and ion channels in compartments of the target neurons, as well as on their location relative to excitatory and inhibitory synaptic sites. Thus, the question arises of how the precise subcellular location of these molecules can be determined at high resolution.

Advanced high-resolution immunocytochemical methods

For this purpose the following advanced high-resolution immunocytochemical methods have been widely used: (i) preembedding immunogold, (ii) postembedding immunogold, and (iii) sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL) techniques. (i) In case of the preembedding immunogold method, an 0.8 nm or a 1.4 nm gold particle is coupled to the secondary antibodies in order to facilitate proper penetration. Silver intensification of the gold particles is subsequently carried out to produce a detectable particle size.

This method produces non-diffusible labels, thus the precise site of the reaction and the location of the protein at extra- and perisynaptic sites can be determined. Synaptic proteins, however, cannot be detected using this method, most likely due to the inaccessibility of the epitopes in the synaptic specializations of fixed tissues [2]. (ii) The postembedding immunogold method overcomes the problems of pre-embedding technique by reacting immunochemicals with the antigens exposed on the surface of the ultrathin sections and then detecting synaptic proteins with the same sensititvity as that for non-synaptic molecules. This also improves quantitative evaluation of the protein densities.

However, in resin-embedded sections substantial proportions of proteins are buried and therefore not accessible for antibodies, limiting the detection sensitivity of this technique [2]. (iii) In SDS-FRL, the brain tissue is frozen with a high-pressure freezing machine (Leica EM HPM100) then frozen samples are freeze-fractured in a replica machine (Leica EM BAF060). Proteins are allocated to either the protoplasmic faces (P-faces) or the exoplasmic faces (E-faces) of plasma membranes. Molecules are immobilized with a thin layer of carbon (3–5 nm) followed by a further coating with a 2-nm-thick platinum/carbon layer for shadowing the membrane faces and then this material is strengthened with a 15–20 nm thick carbon deposit [3]. The SDS-FRL technique has two major advantages compared to conventional immunogold methods.

First, the sensitivity of the SDS-FRL is considerably higher than that of the pre- and postembedding techniques, because membrane proteins are exposed on the two-dimensional surface of the replica (Figure 1), making them readily accessible to immunoreagents. In addition, epitopes are denaturated by SDS, allowing antibodies known to be suitable for immunoblot analysis to react similarly with proteins immobilized on the replica membrane. Second, synaptic and extrasynaptic proteins can simultaneously be visualized and quantification of immunogold density in membrane segments can be achieved.

Fig. 1: Distribution and colocalization of GABA (B1) and Kir3.2 in dendrites of hippocampal cells as revealed by the SDS-digested freezefracture replica labeling technique. A, Immunoparticles for the GABA (B1) subunit were found in clusters (arrows) over the surface of dendritic shaft (Den) and spine (s) of a putative pyramidal cell. B, C, Double and triple immunogold labeling for Kir3.2 (5 nm particles; double arrows), GABA (B1) (10 nm; arrows), and PSD-95 (15 nm in C) revealed that the two proteins co-clustered in dendritic spines of pyramidal cells (B and C) and associated to the site of glutamatergic synapses indicated by immunoreactivity for PSD-95 (C). Scale bars, 200 nm.

This immunocytochemical method, similarly to others, has limitations. First, the identification of labeled morphological structures is difficult, therefore, it is necessary to use marker proteins to facilitate the identification of fractured membranes [6]. Second, the separation of membrane proteins to P-face or E-face is unpredictable: some proteins are preferentially allocated to either the P-face, such as GABA (B1), Kir3.2 [4] or the E-face, such as AMPA receptors [5], whereas others, like gluRδ2 [6] are localized to both faces. Thus, for quantitative studies, the allocation of the molecules should carefully be examined.

Taken together, these three immunocytochemical techniques provide complementary information about the cellular and subcellular distribution of proteins and are widely used for high-resolution qualitative and quantitative analysis of receptor and ion channel localization and colocalization in post- and presynaptic compartments of neurons.